Fish cell lines as a tool for the ecotoxicity assessment and ranking of engineered nanomaterials.

Risk assessment of engineered nanomaterials (ENMs) is being hindered by the sheer production volume of these materials. In this regard, the grouping and ranking of ENMs appears as a promising strategy. Here we sought to evaluate the usefulness of in vitro systems based on fish cell lines for ranking a set of ENMs on the basis of their cytotoxicity. We used the topminnow (Poeciliopsis lucida) liver cell line (PLHC-1) and the rainbow trout (Oncorhynchus mykiss) fibroblast-like gonadal cell line (RTG-2). ENMs were obtained from the EU Joint Research Centre repository. The size frequency distribution of ENM suspensions in cell culture media was characterized. Cytotoxicity was evaluated after 24 h of exposure. PLHC-1 cells exhibited higher sensitivity to the ENMs than RTG-2 cells. ZnO-NM was found to exert toxicity mainly by altering lysosome function and metabolic activity, while multi-walled carbon nanotubes (MWCNTs) caused plasma membrane disruption at high concentrations. The hazard ranking for toxicity (ZnO-NM > MWCNT ≥ CeO2-NM = SiO2-NM) was inversely related to the ranking in size detected in culture medium. Our findings reveal the suitability of fish cell lines for establishing hazard rankings of ENMs in the framework of integrated approaches to testing and assessment.

Thyroid signaling in immune organs and cells of the teleost fish rainbow trout (Oncorhynchus mykiss).

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout (Oncorhynchus mykiss) express both thyroid receptor α (THRA) and ß (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. In vivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells (cd4, cd8a, trb), B lymphocytes (mIgM) and macrophages (csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones.

Non-destructive multibiomarker approach in European quail (Coturnix coturnix coturnix) exposed to the herbicide atrazine.

The effect of orally administered atrazine (25 or 100 mg/kg on days 0, 5, and 10 of the experiment) was studied in European quail (Coturnix coturnix coturnix) on four non-destructive biomarkers: fecal porphyrins, blood glutathione-S-transferase, glutathione reductase, reduced glutathione, and malondialdehyde (MDA). Uroporphyrin I (UPI) and coproporphyrins I and III (CPIII) were the main porphyrins detected in feces. The lowest dose of ATZ caused a significant (P < 0.05) increase in UPI and CPIII at day 5, and the highest dose of ATZ caused an induction of CPI and a significant (P < 0.05) decrease in MDA levels at day 30.

Applicability of OECD TG 201, 202, 203 for the aquatic toxicity testing and assessment of 2D Graphene material nanoforms to meet regulatory needs.

Tests using algae and/or cyanobacteria, invertebrates (crustaceans) and fish form the basic elements of an ecotoxicological assessment in a number of regulations, in particular for classification of a substance as hazardous or not to the aquatic environment according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS-CLP) (GHS, 2022) and the REACH regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals, EC, 2006). Standardised test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) are available to address the regulatory relevant endpoints of growth inhibition in algae and cyanobacteria (TG 201), acute toxicity to invertebrates (TG 202), and acute toxicity in fish (TG 203). Applying these existing OECD TGs for testing two dimensional (2D) graphene nanoforms may require more attention, additional considerations and/or adaptations of the protocols, because graphene materials are often problematic to test due to their unique attributes. In this review a critical analysis of all existing studies and approaches to testing used has been performed in order to comment on the current state of the science on testing and the overall ecotoxicity of 2D graphene materials. Focusing on the specific tests and available guidance's, a complete evaluation of aquatic toxicity testing for hazard classification of 2D graphene materials, as well as the use of alternative tests in an integrated approach to testing and assessment, has been made. This information is essential to ensure future assessments generate meaningful data that will fulfil regulatory requirements for the safe use of this "wonder" material.

In vitro dose-response effects of poly(amidoamine) dendrimers [amino-terminated and surface-modified with N-(2-hydroxydodecyl) groups] and quantitative determination by a liquid chromatography-hybrid quadrupole/time-of-flight mass spectrometry based method.

This article presents a dose-response study of the effects of two types of third-generation (G3) and fourth-generation poly(amidoamine) (PAMAM) dendrimers on two cell lines (RTG-2 and H4IIE) by in vitro cytotoxicity assays with 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. We particularly investigated the potential cytotoxic effect of positive surface charge, which a cationic amino-terminated PAMAM dendrimer can display, on the marked ability of PAMAM dendrimers to cross the cell membrane compared with PAMAM dendrimers functionalized with chains of N-(2-hydroxydodecyl). Quantification of dose-response effects was performed by use of mass spectrometry analysis. The analytical method using liquid chromatography-hybrid quadrupole/time-of-flight mass spectrometry that we developed allowed characterization of defective dendrimers instead of "ideal structures." Identification was based on accurate mass measurement, assignment of elemental composition, and the fully resolved (13)C/(12)C isotopic clusters of the multiply charged ions of PAMAM dendrimers. Validation of the liquid chromatography-mass spectrometry method made possible reliable and reproducible quantification of the extracellular and intracellular concentration of dendrimers at a micromolar level (limits of detection from 0.14 to 1.34 µM and from 0.43 to 1.82 µM in standard and culture medium, respectively). A higher cytotoxicity was found with the H4IIE cell line for surface-modified PAMAM dendrimers. The LDH assay was significantly more sensitive than the MTT and NRU assays, with half-maximal inhibitory concentrations (IC(50)) of 12.96 and 38.31 µg mL(-1) for surface-modified G3 and G4 dendrimers, respectively. No cytotoxic effects, in terms of IC(50), of amino-terminated PAMAM dendrimers were observed on both H4IIE and RTG-2 cells when the concentration was below 500 µg mL(-1) for G3 and G4 dendrimers.

Differences in the induction of cyp1A and related genes in cultured rainbow trout Oncorhynchus mykiss. Additional considerations for the use of EROD activity as a biomarker.

Two rainbow trout Oncorhynchus mykiss fish farms were repeatedly sampled in order to observe the variability of ethoxyresorufin-O-deethylase (EROD) activity and of related genes in the liver. Fish coming from fish farm A exhibited EROD levels that could be considered as basal according to the scientific literature, however, EROD activity in fish coming from fish farm B was significantly increased. This was accompanied by augmented aryl hydrocarbon receptor (ahr) and cytochrome P4501A (cyp1A) messenger RNA expression and reduced oestrogen receptor (er) and vitellogenin (vtg) transcription. Only sediment extracts from the entry channel of fish farm B induced EROD activity in O. mykiss cultured cells, however, this induction could not be explained by the levels of polyaromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) measured in the sediments. The results of this study point out that O. mykiss cultured in fish farms could be used as sentinels for indication of pollution. In this particular work, however, no conclusive evidence has been found for a relationship between the presence of PAHs and PCBs and the observed EROD induction.

Short and long-term effects of nanobiomaterials in fish cell lines. Applicability of RTgill-W1.

Nanobiomaterials (NBMs) are nanostructured materials for biomedical applications that can reach aquatic organisms. The short and long-term effects of these emerging contaminants are unknown in fish. The RTgill-W1 cell line has been proposed as a model to predict the acute toxicity of chemicals to fish (OECD Test Guideline nº 249). We assessed the applicability of this cell line to study the short and long-term toxicity of 15 NBMs based on hydroxyapatites (HA), lipid (LSNP/LNP), gold, iron oxide, carbon, poly l-Lactide acid (PLLA) fibers with Ag and poly (lactide-co-glycolide) acid. Two more rainbow trout cell lines (RTL-W1, from liver, and RTS-11, from spleen) were exposed, to identify possible sensitivity differences among cells. Exposures to a range of concentrations (0.78-100 µg/mL) lasted for 24 h. Additionally, the RTgill-W1 was used to perform long-term (28 d exposure) and recovery (14 d exposure/14 d recovery) assays. Cells were exposed to the 24 h-IC20 and/or to 100 µg/mL. A triple cytotoxicity assay was conducted. After 24 h, only PLLA Fibers-Ag showed cytotoxicity (IC50 < 100 µg/mL). However, the NBMs in general provoked concentration-dependent effects after long-term exposures, except the LSNPs. A recovery of viability was only observed for AuNPs, AuNRods, Fe3O4PEG-PLGA, MgHA-Collag_Scaffolds, Ti-HA and TiHA-Alg NPs.These results evidenced the need to test the long-term toxicity of NBMs and showed differences in cytotoxicity probably associated to different mechanisms of toxic action. The RTgill-W1 was useful to screen short and long-term toxicities of NBMs and appears as a promiseful model to assess possible toxicity of NBMs in fish.

Fish cell lines as screening tools to predict acute toxicity to fish of biocidal active substances and their relevant environmental metabolites.

Biocidal substances and their environmental relevant metabolites are highly toxic for fish. However, an important scarcity of toxicity data for metabolites is recognised. This article provides new data about the toxicity to fish of these compounds and evaluates the potential use of fish cell lines as screening tools to assess the acute toxicity of these compounds in fish. To this aim, acute toxicity of 7 substances was tested in Oncorhynchus mykiss (OECD TG203) and cytotoxicity of 16 substances was assessed in fish cell lines from two species; Poeciliopsis lucida (PLHC-1) and O. mykiss (RTH-149, RTG-2 and RTgill-W1) performing three cytotoxicity tests: Alamar-Blue, 5-carboxyfluorescein diacetate, acetoxymethyl ester and Neutral Red Uptake. Additionally, in vitro and in vivo data from the LIFE-COMBASE database were included in a dataset finally comprising 33 biocides and 14 metabolites. Hazard data were categorized into 4 toxicity groups, according to the intervals established in Regulation (EC) 1272/2008. Finally, the Spearman correlation test was performed and coincidences between in vitro-in vivo data established. In vitro and in vivo results revealed a high positive correlation, with a complete coincidence for 56.5% of the substances, a 2% of false positives (non-toxic in vivo) and a 13% of false negatives (toxic in vivo) for the 4 toxicity categories. However, when results were grouped in toxic or non-toxic coincidence was obtained for 85% of the substances. In conclusion, although fish denote a greater sensitivity, the use of at least two fish cell lines and three cytotoxicity endpoints appear to be valid approaches for fish acute toxicity screening of biocides and their metabolites.

Cytotoxicity against fish and mammalian cell lines and endocrine activity of the mycotoxins beauvericin, deoxynivalenol and ochratoxin-A.

The toxicity of mycotoxins is well recognized in mammals, but their effects on fish have received less attention. Moreover, in the last years several studies have reported that some mycotoxins may act as endocrine disruptors. The aim of this study was to determine the cytotoxic effects and endocrine activities of three mycotoxins: beauvericin, deoxynivalenol and ochratoxin-A. Cytotoxicity in two fish hepatoma and one mammalian hepatoma cell lines was determined by the AlamarBlue, Neutral Red Uptake and CFDA-AM assays. For the assessment of androgenic, estrogenic and thyroidal agonistic/antagonistic effects three cell lines stably expressing luciferase as reporter gene under the control of hormone receptors were used. Results showed that both fish and mammalian cell lines were very sensitive to the mycotoxins tested. OTA was the least toxic mycotoxin and DON and BEA showed similar acute toxicity. None of the three mycotoxins tested presented agonistic effects at the receptors studied, but all of them showed strong antagonistic effect at the thyroid receptor. BEA showed a weak antagonistic effect at the androgen receptor and OTA produced a biphasic dose-response curve at the estrogen receptor. The data obtained in this work are of high interest for aquaculture industries and for regulators.

Is prostatic biopsy as safe as we think? Epidural abscess following prostatic biopsy.

Spinal epidural abscess is an infectious disorder with high morbidity and mortality rates, which is often associated with delayed diagnosis. We report a case of a 73-year-old man with cervical pyogenic spondylodiscitis complicated with epidural abscess following a prostatic biopsy. Clinical presentation included fever, malaise, neck rigidity in all axes, minor paresis of the right arm, and gait ataxia. A cervical vertebral magnetic resonance imaging (MRI) scan showed pyogenic spondylodiscitis with an epidural abscess. Blood, urine, and cerebrospinal fluid cultures were sterile. The patient was treated with intravenous vancomycin, metronidazole, and ceftazidime for 4 weeks, and was discharged from the hospital and treated with oral cloxacillin, metronidazole, and cefixime for another 2 weeks. His neurological symptoms disappeared completely, and he walked normally, without support. It is important for clinicians to be alert to symptoms accompanying back pain following a prostatic biopsy and to consider the possibility of a diagnosis of spinal abscess.

Development of a new tool for the long term in vitro ecotoxicity testing of nanomaterials using a rainbow-trout cell line (RTL-W1).

The current wide use of manufactured nanomaterials (MNs) is leading to the release of nanoparticles (NPs) to water bodies. Aquatic organisms, including fish, are exposed to low concentrations of NPs for long periods of time being necessary to develop laboratory toxicity tests reflecting realistic conditions. Additionally, today there is a demand of in vitro assays respecting the 3Rs principle. Thus, the main aim of this work was to stablish an in vitro tool for the assessment of long-term NPs ecotoxicity. Considering the key role of liver in detoxification, a rainbow trout liver cell line, RTL-W1, was used. CuO NPs were chosen to validate this tool taking into account their important production level. Cells were exposed for 21 days to 25 or 100 µg CuO NPs/ml. Every seven days cells were split and one fourth of them transferred to a new plate with appropriate concentrations of NPs in culture medium. Lower concentrations of CuO NPs did not cause any deleterious effect, whereas higher concentrations led to significant mortality after 14 days and to the intracellular accumulation of Cu particles. Identical results were observed in cells exposed to CuSO4 at the same Cu concentrations. Therefore, the observed toxic effects might be mainly due to Cu2+ ions.

Determining the presence of chemicals with suspected endocrine activity in drinking water from the Madrid region (Spain) and assessment of their estrogenic, androgenic and thyroidal activities.

Endocrine disruptors (EDs) are natural or man-made chemicals that can affect the health of organisms by interfering with their normal hormonal functions. Many of these substances can cause their effects at very low doses and, considering the key role played by the endocrine system on development, organisms in early phases of growth (foetal, childhood, puberty) are especially sensitive to the action of EDs. In addition, when combined, they can show additive, antagonistic and synergistic activities. Taking all this into account it is essential to determine the presence of this kind of compounds in drinking water. Thus the main aim of the present study was to monitor the presence of substances with suspected or known endocrine activity in drinking water of the Madrid Region (MR) (Central Spain) and determine possible estrogenic, androgenic, or thyroidal activities. Water samples were collected at different times from a number of supply points that received water from reservoirs or rivers. The sampling point with the highest concentration of the analysed substances (up to 30 compounds) was DW1 (1203 ng L-1). This sampling point receives water from a drinking water treatment plant (DWTP) that serves the population from the south of the MR with treated water from the Tajuña River. DW2 was the second point with the highest concentration of the analysed substances (1021 ng L-1). DW2 receives water from one of the reservoirs in the north of the MR. The highest daily concentrations detected corresponded to the flame retardant Tris (2-chloroethyl)phosphate (TCEP) (266.55 ng L-1) and to the nonylphenol diethoxylate (188.57 ng L-1) at points DW1 and DW4, respectively, both of which are supplied with treated river water. None of the water samples exhibited androgenic, oestrogenic, or thyroidal activities in in vitro assays based on cells stably transfected with the receptors of interest and luciferase as reporter gene. These results demonstrate that water quality in the MR is high and does not present a health risk for the population, although the concentrations of some substances justify the need for local authorities to continually monitor the presence of these contaminants in order to implement any corrective measures if necessary.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study.

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Effects of a silver nanomaterial on cellular organelles and time course of oxidative stress in a fish cell line (PLHC-1).

Among the nanomaterials currently in commercial products, those based on silver are the most used, and so there is a high probability that silver nanoparticles (AgNPs) will be released into aquatic environments where they could adversely affect aquatic organisms, including fish. Taking this into account, the aim of the present work was to characterize in depth the mechanisms underlying the toxic action of AgNPs using fish cell lines, determining specifically the contribution of alterations in cellular structures and oxidative stress time course to the cytotoxicity of AgNPs. Since liver plays a key role in detoxification, the hepatoma cell line PLHC-1 was used. Exposure to AgNPs (NM-300K, obtained from the Joint Research Centre Repository) caused alterations at the lysosomal and mitochondrial levels at lower concentrations than those that disrupted plasma membrane (evaluated by means of neutral red, alamarBlue, and 5-carboxyfluorescein diacetate, acetoxymethyl ester assays respectively). AgNO3, used as a control Ag+ ion source, produced similar cytotoxic effects but at lower concentrations than AgNPs. Both silver forms caused oxidative disruption but the initial response was delayed in AgNPs until 6h of exposure. Transmission electron microscopy analysis also evidenced the disruption of mitochondrial structures in cells exposed to cytotoxic concentrations of both forms of silver. At non-cytotoxic concentrations, AgNPs were detected inside the nucleoli and mitochondria, thereby pointing to long-term effects. The present work evidences the mutual interaction between the induction of oxidative stress and the alterations of cellular structures, particularly mitochondria, as cytotoxicity mechanisms not exclusively associated to NPs.

Cytochrome P4501A induction caused by the imidazole derivative Prochloraz in a rainbow trout cell line.

A variety of aquatic pollutants are able to induce cytochrome P4501A (CYP1A) in fish by ligand binding to the aryl hydrocarbon receptor (AhR). High-affinity AhR ligands are planar aromatic polycyclic molecules such as the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present work investigates the ability of the imidazole derivative, Prochloraz (PRO), to induce CYP1A. Computational studies on the molecular structure of PRO indicated that it is highly unlikely for PRO to have both aromatic rings of the molecule, i.e. the imidazole and the benzene ring, in the same plane. Thus, the possible conformers do not take planar structures, in contrast to the typically planar AhR ligands. Experimentally, the capability of PRO to induce CYP1A was assessed using the rainbow trout liver cell line, RTL-W1, as in vitro model. PRO increased in a concentration-dependent way the catalytic activity of CYP1A (determined as 7-ethoxyresorufin-O-deethylase, EROD, activity) in RTL-W1 cells. The potency of PRO was lower than that of a reference AhR-ligand, beta-naphthoflavone (betaNF). In addition to the catalytic level, PRO activated CYP1A also at the transcriptional level as determined by RT-PCR analysis of CYP1A mRNA. These results indicate that PRO, although its structure is not corresponding to the typical features of CYP1A-inducing AhR ligands, still is able to activate CYP1A expression.

Organochlorine compounds in liver and concentrations of vitellogenin and 17beta-estradiol in plasma of sea bass fed with a commercial or with a natural diet.

Results from previous experiments directed to determine the effect of different nutritional factors or the effect of xenobiotics on hormonal control of reproduction, lead to the hypothesis that hormonal perturbations repeatedly observed in sea bass (Dicentrarchus labrax) broodstock feeding commercial diets could have been caused by the presence of aryl hydrocarbon receptor (AhR) ligands, such as dioxins, furans and polychlorinated biphenyls (PCBs) in the diet. To evaluate this hypothesis, dioxins and related compounds were analysed in liver of female sea bass fed with a commercial or with a natural diet consisting of trash fish (bogue, Boops boops), and concentrations of vitellogenin (VTG) and 17beta-estradiol (E2) were determined in plasma obtained previously in monthly samplings of these animals. As observed in other experiments, females fed with a commercial diet exhibited lower VTG and higher E2 plasma levels than females fed with the natural diet. In liver, sea bass fed with the commercial diet exhibited a profile clearly dominated by high-chlorinated dioxins while in fish fed with the natural diet this profile was dominated by low chlorinated furans. However, typical AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin showed no differences between groups or, as is the case of planar PCBs, showed higher concentrations in the liver of fish fed with the natural diet. These results do not permit to explain the observed hormonal alterations by a possible antiestrogenic effect caused by dioxins and related compounds.

Do gonadotrophin-releasing hormone neurons express estrogen receptors in the rainbow trout? A double immunohistochemical study.

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distributions of estrogen receptor-immunoreactive cells and gonadotrophin-releasing hormone-immunoreactive neurons on the same sections of the brains of adult male and female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the ventral and lateral telencephalon, the preoptic region, the mediobasal hypothalamus, and the ventromedial thalamic nucleus. Gonadotrophin-releasing hormone-immunoreactive perikarya were detected in the olfactory bulbs, the ventral telencephalon, the preoptic area, and the mediobasal hypothalamus. Double-staining studies showed that, although some estrogen receptor-positive cells were in close proximity to gonadotrophin-releasing hormone-immunoreactive perikarya, careful examination of 550 gonadotrophin-releasing hormone-positive cells from five adult females and two adult males failed to demonstrate any evidence that gonadotrophin-releasing hormone neurons coexpress estrogen receptor in the brain of the rainbow trout. The present study provides, for the first time in teleosts, morphological evidence that gonadotrophin-releasing hormone neurons do not represent major direct targets for estradiol, suggesting that the positive feedback effects of estradiol onto the gonadotrophin-releasing hormone system are likely to be conveyed via other cell populations.

Estrogen-mediated suppression of cytochrome P4501A (CYP1A) expression in rainbow trout hepatocytes: role of estrogen receptor.

Hepatic CYP1A expression in fish can be modulated by the female sex hormone, 17beta-estradiol (E2), however neither the mechanism of E2 suppression of CYP1A nor the capacity for hormonal regulation to overcome CYP1A induction by xenobiotics are known. The present study investigates for the first time in fish if the estrogen receptor (ER) is involved in the suppressive action of E2 on CYP1A gene expression. The study further examines, if the E2 effect is able to overcome xenobiotic induction of CYP1A. As experimental model, in vitro cultures of rainbow trout, Oncorhynchus mykiss, hepatocytes were used. The effect of E2 on CYP1A was assessed by measuring the CYP1A-associated 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity, and CYP1A mRNA contents. E2 at non-cytotoxic concentrations caused a significant time- and concentration-dependent decline of basal but not of induced hepatic EROD activities. The inhibitory action of E2 on basal CYP1A was also evident at the mRNA level. The presence of the ER antagonist tamoxifen abolished the inhibitory action of E2 on CYP1A expression. The results from these in vitro experiments provide evidence (a) that the ER is involved in the suppressive action of E2 on CYP1A, and (b) that E2 inhibitory action does not overcome xenobiotic induction of CYP1A.

Antiestrogenicity of beta-naphthoflavone and PAHs in cultured rainbow trout hepatocytes: evidence for a role of the arylhydrocarbon receptor.

The aims of the present study were to assess, (1) if polyaromatic hydrocarbons (PAHs) are able to inhibit estradiol-regulated vitellogenin synthesis in fish; and (2) if this antiestrogenic activity is mediated through the binding of PAHs to the arylhydrocarbon receptor (AhR). Cultured liver cells of rainbow trout, Oncorhynchus mykiss, were co-exposed to PAHs and 17beta-estradiol (E2), and the resulting effects on induction of AhR-regulated 7-ethoxyresorufin-O-deethylase (EROD) activity and on E2-regulated vitellogenesis were investigated. The following test compounds were compared: the PAH 3-methylcholanthrene (3MC), which is a strong EROD inducer, the PAH anthracene (ANT), which is not an inducer of EROD activity, and the model EROD inducer, beta-naphthoflavone (betaNF). 3MC and betaNF led to significant decreases of E2-triggered hepatocellular VTG synthesis, whereas ANT exerted no antiestrogenic activity. The rank order of the antiestrogenic activity of the test substances agreed with their EROD-inducing potency suggesting that their antiestrogenicity might be mediated through the AhR. Further evidence for this assumption comes from the observation that inhibitors such as alpha-naphthoflavone which interferes with ligand-AhR binding, and 8-methoxypsoralen (8MP), which prevents binding of the occupied AhR to responsive DNA elements, clearly reduced the antiestrogenic effects of the xenobiotics. Furthermore, from the comparison of estradiol concentrations in media of liver cells exposed to the CYP 1A-inducing agents and in media of control cells it is unlikely that the observed antiestrogenic effects were caused by an enhanced E2 catabolism. In conclusion, the results from this study indicate that, (1) AhR-binding PAHs possess an antiestrogenic activity; and (2) that the antiestrogenic activity is mediated through the AhR.

Modulation of trout 7-ethoxyresorufin-O-deethylase (EROD) activity by estradiol and octylphenol.

Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.